Abstract
BackgroundPluripotent, Lin–/CD45–/Sca-1+ very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin–/CD45+/Sca-1+ hematopoietic stem cells (HSCs) get eliminated. In the present study, we provide further evidence for the existence of VSELs in mouse BM and have also examined the effects of a chemotherapeutic agent (5-fluorouracil (5-FU)) and gonadotropin hormone (follicle-stimulating hormone (FSH)) on BM stem/progenitor cells.MethodsVSELs and HSCs were characterized in intact BM. Swiss mice were injected with 5-FU (150 mg/kg) and sacrificed on 2, 4, and 10 days (D2, D4, and D10) post treatment to examine changes in BM histology and effects on VSELs and HSCs by a multiparametric approach. The effect of FSH (5 IU) administered 48 h after 5-FU treatment was also studied. Bromodeoxyuridine (BrdU) incorporation, cell cycle analysis, and colony-forming unit (CFU) assay were carried out to understand the functional potential of stem/progenitor cells towards regeneration of chemoablated marrow.ResultsNuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN–/CD45– VSELs and slightly larger LIN–/CD45+ HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10. Nuclear OCT-4 and PCNA-positive VSELs were detected in chemoablated bone sections near the endosteal region. VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna). Cells that survived 5-FU formed colonies in vitro. Both VSELs and HSCs expressed FSH receptors and FSH treatment enhanced hematopoietic recovery by 72 h.ConclusionBoth VSELs and HSCs were activated in response to the stress created by 5-FU and FSH enhanced hematopoietic recovery by at least 72 h in 5-FU-treated mice. VSELs are the most primitive pluripotent stem cells in BM that self-renew and give rise to HSCs under stress, and HSCs further divide rapidly and differentiate to maintain homeostasis. The study provides a novel insight into basic hematopoiesis and has clinical relevance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0311-6) contains supplementary material, which is available to authorized users.
Highlights
Pluripotent, Lin–/CD45–/Sca-1+ very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin–/CD45+/Sca-1+ hematopoietic stem cells (HSCs) get eliminated
Mouse BM harbors pluripotent VSELs Earlier reports [6, 53] and our initial immunofluorescence studies on cell smears of mice bone marrow confirmed the presence of rare, small, spherical cells with high nucleo-cytoplasmic ratio expressing pluripotent stem cell markers including nuclear OCT-4A and SOX2 and cell-surface SCA-1 and SSEA-1 (Fig. 1a)
A similar strategy was used to determine the percentage of small cells expressing OCT-4A and SSEA-1 and it was observed that the percentage of LIN–/CD45– cells co-expressing these markers was almost similar to SCA-1+ VSELs (Additional file 1: Table S3)
Summary
Pluripotent, Lin–/CD45–/Sca-1+ very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin–/CD45+/Sca-1+ hematopoietic stem cells (HSCs) get eliminated. In 2006, a rare population of these cells expressing pluripotent markers Oct-4 and Nanog was identified and well characterized in the bone marrow [6] Over the decade these LIN–/CD45–/SCA-1+ in mice and CD133+ in humans, very small embryonic-like stem cells (VSELs) have been described in various murine and human adult tissues including bone marrow [7, 8], cord blood [9,10,11], testis [12, 13], ovary [14, 15], uterus [16], pancreas [17], and other organs [18, 19]. The role of VSELs in hematopoiesis in vivo either in steady-state conditions or in response to bone marrow injury has not been studied extensively
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